Rocky Mountain Spotted Fever (RMSF) is a severe human disease caused by the obligate intracellular bacterium, Rickettsia rickettsii. Presently, there is no vaccine available for RMSF, and little information is available on the molecular/genetic biology of R. rickettsii or on virulence determinants. The aims of the work outlined in this proposal are to:1) Evaluate the use of a R. rickettsii protein immunogen, cloned and expressed in Escherichia coli, as a sub-unit vaccine for RMSF; 2) Study the temporal regulation of R. rickettsii gene expression and protein production, and also to study these processes in response to different growth temperatures; 3)To use a molecular genetics approach to identify and characterize virulence determinants of R. rickettsii. To accomplish these goals I intend to: 1) Obtain a high level of expression of a cloned gene encoding a major R. rickettsii protein immunogen, purify the cloned product and test its ability to immunize guinea pigs against challenge with viable R. rickettsii; 2) Examine the expression of R. rickettsii genes by monitoring mRNA and protein production; 3) Utilize a molecular genetics approach to identify components of R. rickettsii responsible for attachment to host cells and subsequent invasion, clone and characterize the genes encoding these components, and study their regulation. Virulence determinants will be mutagenized and the abilities of the mutants to produce disease will be assessed. Because the understanding of aspects of the molecular biology and genetics of R. rickettsii is in the infantile stage, I consider some of the experiments outlined in this proposal to be pioneering but highly significant. The data obtained from these studies will be useful in the eventual development of a vaccine suitable for use in humans, and in an understanding, at the molecular level, of the components/events in a R. rickettsii infection.